Costimulatory b7-h1 in renal cell carcinoma patients: indicator of tumor aggressiveness and potential therapeutic target

ABSTRACT

The invention features methods of diagnosis by assessing B7-H1 expression in a tissue from a subject that has, or is suspected of having, cancer, methods of treatment with agents that interfere with B7-H1-receptor interaction, methods of selecting candidate subjects likely to benefit from cancer immunotherapy, and methods of inhibiting expression of B7-H1.

This application is a Continuation of U.S. patent application Ser. No.13/012,063, filed Jan. 24, 2011, which is a Continuation of U.S. patentapplication Ser. No. 11/245,713, now U.S. Pat. No. 7,892,540, whichclaims the benefit of U.S. Provisional Application No. 60/616,590, filedOct. 6, 2004, and U.S. Provisional Application 60/642,794, filed Jan.11, 2005. The disclosures of all prior applications are expresslyincorporated herein by reference in their entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

Funding for the work described herein was provided by the federalgovernment under grant awarded by the National Institute of Health. Thefederal government has certain rights in the invention.

TECHNICAL FIELD

This invention relates to immune molecules expressed in cancer tissue,and more particularly to evaluating the expression of immune moleculesin tumor cells and tumor-infiltrating leukocytes.

BACKGROUND

An important determinant for the initiation and progression of cancer isthe ability of cancer cells to evade the host's immune system. Thepresence in cancer tissue of, for example, inadequate, inappropriate, orinhibitory immune molecules can restrict the host's ability to generateimmune responses to the cancer.

The disclosures of U.S. Pat. No. 6,803,192 and co-pending U.S.application Ser. Nos. 09/649,108; 10/127,282; and Ser. No. 10/719,477;and International Application No. US/02/32364 are incorporated herein byreference in their entirety.

SUMMARY

The invention is based in part on the finding that in renal cellcarcinoma (RCC) patients the risk of death is proportional to the numberof tumor cells, and/or leukocytes in the tumor, expressing theco-stimulatory human glycoprotein B7-H1. As used herein, the term“B7-H1” refers to B7-H1 from any mammalian species and the term “hB7-H1”refers to human B7-H1. Further details on B7-H1 polypeptides and nucleicacids are provided in U.S. Pat. No. 6,803,192 and co-pending U.S.application Ser. No. 09/649,108, the disclosures of which areincorporated herein by reference in their entirety.

The invention provides methods of diagnosing subjects having, or thatare likely to develop, cancer of a tissue based on the expression ofB7-H1 by cells of the cancer tissue, methods of predicting success ofimmunotherapy, methods of prognosis, and methods of treatment.Leukocytes in a tumor are sometimes referred to herein as“tumor-infiltrating leukocytes” or “leukocytes infiltrating a/thetumor.”

More specifically, the invention provides a method of diagnosis ofcancer in a subject. The method involves: (a) providing a tissue samplefrom a subject suspected of having, or likely to develop, cancer of thetissue, wherein the sample contains test cells, the test cells beingcells of the tissue or leukocytes infiltrating the tissue; and (b)assessing whether the test cells express B7-H1, wherein expression bysome or all of the test cells is an indication that the subject hascancer.

The assessment of B7-H1 expression can be performed by the detection ofB7-H1 polypeptide or mRNA. B7-H1 polypeptide can be detected, forexample, by contacting the tissue sample, or test cells contained in thetissue sample, with an antibody that binds to the B7-H1 polypeptide.Suitable methods for detection of B7-H1 polypeptide can include, withoutlimitation, fluorescence flow cytometry (FFC) or immunohistology. B7-H1mRNA can be detected, for example, by contacting the tissue sample witha nucleic acid probe that hybridizes to the B7-H1 mRNA (e.g., such by insitu hybridization) or by reverse transcriptase-polymerase chainreaction. The tissue can be tissue of any organ or anatomical system,and can include, without limitation, lung, epithelial, connective,vascular, muscle, neural, skeletal, lymphatic, prostate, cervical,breast, spleen, gastric, intestinal, oral, esophageal, uterine, ovarian,or testicular tissue. The tissue can also be renal tissue. The subjectcan be a mammal, such as, for example, a human.

Another aspect of the invention is a method of identifying a candidatefor immunotherapy. This method involves: (a) providing a tissue samplefrom a subject with cancer of the tissue, wherein the tissue samplecontains test cells, the test cells being cancer cells ortumor-infiltrating leukocytes; and (b) assessing the level of test cellsin the tissue sample that express B7-H1, wherein, if B7-H1 expression isnot detected in the test cells or if less than an immuno-inhibitorythreshold level of the test cells express B7-H1, the subject is morelikely to benefit from immunotherapy.

The level of B7-H1 can be assessed by detecting B7-H1 polypeptide ormRNA using, for example, any of the methods described above for methodof diagnosis. The tissue can be tissue of any organ or anatomicalsystem, and can include, without limitation, lung, epithelial,connective, vascular, muscle, neural, skeletal, lymphatic, prostate,cervical, breast, spleen, gastric, intestinal, oral, esophageal,uterine, ovarian, or testicular tissue. The tissue can also be renaltissue. The subject can be a mammal, such as, for example, a human. Thecancer can be any cancer, and includes, for example, renal cellcarcinoma.

In another embodiment, the invention features a method of determiningthe prognosis of a subject with cancer. This method involves: (a)providing a tissue sample from a subject with cancer of the tissue,wherein the tissue sample comprises test cells, the test cells beingcancer cells or tumor-infiltrating leukocytes; and (b) assessing thelevel of test cells in the tissue sample that express B7-H1, wherein, ifa prognostic level, or more than a prognostic level, of the test cellsexpress B7-H1, the subject is more likely to die of the cancer than ifless than a prognostic level of the test cells express B7-H1. Theprognostic level is a predetermined value obtained by performingstatistical clinical analyses known in the art, e.g., those describedherein. The assessment of B7-H1 can be performed by detecting B7-H1polypeptide or B7-H1 mRNA using any of a variety of methods known in theart, including, for example, those listed above for methods of diagnosisand method of immunotherapy. The tissue sample can be of any tissue, andcan include, for example, any of those described above. The subject fromwhich the tissue is provided can be a mammal, e.g., a human.

Yet another aspect of the invention is a method of treatment. The methodinvolves: (a) identifying a subject with cancer, wherein some or allcells of the cancer or some or all tumor-infiltrating leukocytes of thecancer express B7-H1; and (b) delivering to the subject an agent thatinterferes with an interaction between B7-H1 and a receptor for B7-H1.The agent can bind to B7-H1 or to a receptor for B7-H1, e.g., the PD-1receptor. The agent can be an antibody or an antibody fragment (e.g.,Fab′, F(ab′)₂, or single chain Fv (scFv) fragment) that binds to B7-H1or binds to a receptor for B7-H1; soluble B7-H1 or a soluble functionalfragment of B7-H1; a soluble receptor for B7-H1 or a soluble functionalfragment thereof. Whenever it is desired, the agent can be administeredbefore, simultaneous with, or after administration of one or moreimmunomodulatory cytokines, growth factors, or antiangiogenic factors.Examples of such immunomodulatory cytokines, growth factors, andantiangiogenic factors include, without limitation, any of interleukins(L)-1 to 25, interferon-γ (IFN-γ), interferon-α (IFN-α), interferon-β(IFN-β), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α),granulocyte macrophage colony stimulating factor (GM-CSF), granulocytemacrophage colony stimulating factor (G-CSF), endostatin, angiostatin,and thrombospondin. Administrations of the agent and/or the one or moreimmunomodulatory cytokines, growth factors, or antiangiogenic factorscan be systemic (e.g., intravenous) or local, e.g., during surgery bydirect injection or infusion into the tissue that comprises the cells ofthe cancer and/or tumor-infiltrating leukocytes. The cancer can be,without limitation, hematological cancer, neurological cancer, melanoma,breast cancer, lung cancer, head and neck cancer, gastrointestinalcancer, liver cancer, pancreatic cancer, renal cancer, genitourinarycancer, bone cancer, or vascular cancer.

Yet another aspect of the invention is a method of inhibiting theexpression of B7-H1 in a tumor cell or a tumor-infiltrating leukocyte.The method involves: (a) identifying a subject with cancer, the cancercontaining a target cell that expresses B7-H1, the target cell being atumor cell or a tumor-infiltrating leukocyte; and (b) introducing intothe target cell: (i) an antisense oligonucleotide that hybridizes to aB7-H1 transcript, wherein the antisense oligonucleotide inhibits theexpression of B7-H1 in the cell; or (ii) a B7-H1 interference RNA(RNAi). The introducing step can involve administration of the antisenseoligonucleotide or the RNAi to the subject and uptake of theoligonucleotide or the RNAi by the target cell. Alternatively, theintroducing step can involve administering to the subject, and uptake bythe cell of, a nucleic acid comprising a transcriptional regulatoryelement (TRE) operably linked to a nucleotide sequence complementary tothe antisense oligonucleotide, wherein transcription of the nucleotidesequence inside the cell produces the antisense oligonucleotide.Moreover, the introducing step can include administering to the subject,and uptake by the cell of, a nucleic acid: (a) from which sense andanti-sense strands of the RNAi can be transcribed under the direction ofthe TREs; or (b) from which both sense and anti-sense strands of theRNAi can be transcribed under the direction of a single TRE.

The tissue sample can be lung, epithelial, connective, vascular, muscle,neural, skeletal, lymphatic, prostate, cervical, breast, spleen,gastric, intestinal, oral, esophageal, dermal, liver, bladder, thyroid,thymic, adrenal, brain, gallbladder, pancreatic, uterine, ovarian, ortesticular tissue. The tissue can also be renal tissue. The cancer ofthe tissue can be any cancer and includes, e.g., renal cell carcinoma.

The subject can be a mammal and includes, for example, a human, anon-human primate (e.g., a monkey), a horse, a cow (or an ox or bull), apig, a sheep, a goat, a cat, a rabbit, a guinea pig, a hamster, a rat,or a gerbil.

As used herein, “interferes with an interaction between B7-H1 and areceptor for B7-H1” means (a) completely blocks a physical interactionbetween B7-H1 molecule and a receptor for B7-H1 such that there issubstantially no physical interaction between the B7-H1 molecule and thereceptor; or (b) modifies the interaction between the B7-H1 molecule andthe receptor such that the physical interaction either does not delivera signal to the cell that comprises B7-H1, and/or the receptor forB7-H1, or delivers a signal that does not substantially affect theantitumoral activity of the cell.

“Polypeptide” and “protein” are used interchangeably and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification. Polypeptides useful for the inventioninclude variant polypeptides that are identical to correspondingwild-type polypeptides but differ by not more than 50 (e.g., not morethan: 45; 40; 35; 30; 25; 20; 19; 18; 17; 16; 15; 14; 13; 12; 11; 10;nine; eight; seven; six; five; four; three; two; or one) conservativesubstitution(s). All that is required is that the variant polypeptidehas at least 20% (e.g., at least: 25; 30%; 35%; 40%; 45%; 50%; 60%; 70%;80%; 85%; 90%; 93%; 95%; 96%; 97%; 98%; 99%; 99.5%; 99.8%; 99.9%; or100% or more) of the activity of the wild-type polypeptide. Conservativesubstitutions typically include substitutions within the followinggroups: glycine and alanine; valine, isoleucine, and leucine; asparticacid and glutamic acid; asparagine, glutamine, serine, and threonine;lysine, histidine, and arginine; and phenylalanine and tyrosine.

As used herein, “tumor-infiltrating leukocytes” can be T lymphocytes(such as CD8⁺ T lymphocytes and/or CD4⁺ T lymphocytes), B lymphocytes,or other bone marrow-lineage cells including granulocytes (neutrophils,eosinophils, basophils), monocytes, macrophages, dendritic cells (i.e.,interdigitating dendritic cells), histiocytes, and natural killer cells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

Other features and advantages of the invention will be apparent from thefollowing description, from the drawings and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a series of photomicrographs (at a magnification of 400×)showing immunostaining (with an antibody specific for hB7-H1) of: an RCCspecimen with high tumor cell hB7-H1 expression (FIG. 1A); an RCCspecimen with high leukocyte hB7-H1 expression (FIG. 1B); an RCCspecimen with no detectable hB7-H1 expression in either tumor cells orleukocytes (FIG. 1C); and a normal kidney specimen with no detectablehB7-H1 expression in the proximal tubules (FIG. 1D).

FIG. 2 is a series of line graphs showing the associations of hB7-H1expression with death from RCC in 196 subjects from whom the clear cellRCC specimens were obtained for analysis.

FIG. 2A shows the association of tumor hB7-H1 expression with death fromRCC (risk ratio 2.91; 95% CI [Confidence Interval] 1.39-6.13; p=0.005).The cancer-specific survival rates (with standard error [SE] and numberstill at risk indicated in parentheses) at 1, 2, and 3 years followingnephrectomy were: 87.8% (4.1%, 53), 72.3% (6.0%, 30), and 63.2% (7.2%,11), respectively, for patients with specimens that had ≧10% tumorhB7-H1 expression; compared with 93.6% (2.3%, 95), 88.4% (3.4%, 48), and88.4% (3.4%, 19), respectively, for patients with specimens that had<10% tumor hB7-H1 expression.

FIG. 2B shows the association of adjusted score for leukocyte hB7-H1expression with death from RCC (risk ratio 3.58; 95% CI 1.74-7.37;p<0.001). The cancer-specific survival rates (SE, number still at risk)at 1, 2, and 3 years were: 83.5% (6.2%, 26), 63.9% (9.2%, 13), and 53.6%(10.2%, 5), respectively, for patients with specimens that had aleukocyte hB7-H1 expression score ≧100; compared with 93.5% (2.1%, 122),86.2% (3.3%, 65), and 84.8% (3.5%, 25), respectively, for patients withspecimens that had scores <100.

FIG. 2C shows the association of high aggregate intratumoral hB7-H1expression with death from RCC (risk ratio 4.53; 95% CI 1.94-10.56;p<0.001). The cancer-specific survival rates (SE, number still at risk)at 1, 2, and 3 years were: 87.0% (3.8%, 61), 70.0% (5.8%, 32), and 61.9%(6.8%, 13), respectively, for patients with specimens that had highaggregate intratumoral hB7-H1 expression; compared with 94.9% (2.2%,87), 91.9% (3.1%, 46), and 91.9% (3.1%, 17), respectively, for patientswith specimens that had both <10% tumor and <100 leukocyte (lowaggregate intratumoral expression) hB7-H1 expression.

FIG. 3 is a depiction of the amino acid sequence (SEQ ID NO:1) offull-length, immature hB7-H1, i.e., hB7-H1 including a leader peptide ofabout 22 amino acids.

FIG. 4 is a depiction of the nucleotide sequence (SEQ ID NO:2) of cDNAencoding full-length, immature hB7-H1.

FIG. 5 is a depiction of the amino acid sequence (SEQ ID NO:3) offull-length, immature murine B7-H1.

FIG. 6 is a depiction of the nucleotide sequence (SEQ ID NO:4) of cDNAencoding full-length, immature murine B7-H1.

DETAILED DESCRIPTION

The inventors have discovered that renal cell carcinoma (RCC) patientswho have increased levels of tumor cells and/or tumor-infiltratingleukocytes expressing the co-stimulatory glycoprotein hB7-H1 are at anincreased risk of death from the RCC. In addition, elevated levels ofhB7-H1 expressing tumor cells and/or tumor-infiltrating leukocytes wasassociated with more aggressive tumors and this association persistedeven after controlling for traditional predictors of RCC progression,including, for example, tumor, node, metastasis (TNM) stage; primarytumor size; nuclear grade; and histological tumor necrosis.

Expression of B7-H1 in normal, non-activated mammalian cells is largely,if not exclusively, limited to macrophage-lineage cells and provides apotential costimulatory signal source for regulation of T cellactivation. In contrast, aberrant expression of B7-H1 by tumor cells hasbeen implicated in impairment of T cell function and survival, resultingin defective host antitumoral immunity.

The inventors found that human RCC tumors express hB7-H1. In particular,hB7-H1 was found to be expressed by both renal cell carcinoma (RCC)tumors and leukocytes infiltrating RCC tumors. In contrast, proximaltubules of the renal cortex, from which clear cell tumors are believedto arise, failed to express hB7-H1.

Clinical specimens were obtained from 196 patients who were treated withradical nephrectomy or nephron-sparing surgery for unilateral, clearcell RCC between 2000 and 2002 from the Mayo Clinic NephrectomyRegistry. Immunohistological detection and quantification of hB7-H1expression in the specimens revealed that patients whose tumor specimensexhibited high intratumoral expression levels of hB7-H1 (contributed bytumor cells alone, leukocytes alone, or tumor and/or leukocytescombined) had aggressive tumors and were at markedly increased risk ofdeath from RCC.

The combination of increased tumor cell hB7-H1 and tumor-infiltratingleukocyte hB7-H1 (high aggregate intratumoral hB7-H1) was an evenstronger predictor of patient outcome than either hB7-H1-expressingtumor cells or tumor-infiltrating leukocytes alone. High aggregateintratumoral hB7-H1 expression levels were also significantly associatedwith regional lymph node involvement, distant metastases, advancednuclear grade, and the presence of histologic tumor necrosis.

Based on its ability to impair function and survival of activatedtumor-specific T cells, B7-H1, expressed by either tumor cells (e.g.,RCC cells) or infiltrating leukocytes, can contribute to theimmunosuppression that is commonly observed in subjects with cancer(e.g., RCC) and can serve as a critical determinant of the subjects'responses to immunotherapy for management of advanced cancer (e.g.,IL-2, IL-12, IFN-α, vaccination or T-cell adoptive therapy). This raisesthe possibility that administering to cancer patients agents thatinterfere with the interaction of B7-H1 with its receptor (e.g., PD-1)can serve as a method of immunotherapy, particularly in subjects whosehigh level of intratumoral B7-H1 expression previously rendered themunresponsive or nearly unresponsive to other modes of immunotherapy.

These findings provide support for the methods of the invention, whichare described below.

Methods of Diagnosis

The invention provides a method of diagnosing cancer in a subject. Themethod involves: (a) providing a tissue sample from a subject suspectedof having, or likely to develop, cancer of the tissue, the samplecontaining test cells, the test cells being cells of the tissue orleukocytes infiltrating the tissue; and (b) assessing whether the testcells express B7-H1. Expression by some or all of the test cells is anindication that the subject has cancer. Since a wide variety of cancercells express B7-H1 on their surfaces, the methods of the invention areparticularly useful for diagnosing any such cancer. Test cells can thusbe, for example, breast cells, lung cells, colon cells, pancreaticcells, renal cells, stomach cells, liver cells, bone cells,hematological cells (e.g., lymphoid cells, granulocytic cells, monocytesor macrophages), neural tissue cells, melanocytes, ovarian cells,testicular cells, prostate cells, cervical cells, vaginal cells, bladdercells, or any other cells listed herein. Moreover, test cells can beleukocytes present in relevant tissues containing any of theabove-listed test cells. Leukocytes infiltrating the tissue can be Tcells (CD4⁺ T cells and/or CD8⁺ T cells) or B lymphocytes. Suchleukocytes can also be neutrophils, eosinophils, basophils, monocytes,macrophages, histiocytes, or natural killer cells. Subjects can bemammals and include, for example, humans, non-human primates (e.g.,monkeys, baboons, or chimpanzees), horses, cows (or oxen or bulls),pigs, sheep, goats, cats, rabbits, guinea pigs, hamsters, rats, gerbils,or mice.

As described herein, the invention provides a number of diagnosticadvantages and uses. In the methods of the invention, the level of B7-H1polypeptide and/or mRNA can be assessed. The level of B7-H1 is assessedin a tissue sample to diagnose, or to confirm, the presence of cancer inthe subject from whom the tissue is obtained.

Methods of detecting a polypeptide in a tissue sample are known in theart. For example, antibodies (or fragments thereof) that bind to anepitope specific for B7-H1 can be used to assess whether test cells fromthe tissue sample express B7-H1. Such antibodies can be monoclonal orpolyclonal antibodies. In such assays, the antibody itself, or asecondary antibody that binds to it, can be detectably labeled.Alternatively, the antibody can be conjugated with biotin, anddetectably labeled avidin (a polypeptide that binds to biotin) can beused to detect the presence of the biotinylated antibody. Combinationsof these approaches (including “multi-layer sandwich” assays) familiarto those in the art can be used to enhance the sensitivity of themethodologies. Some of these protein-detecting assays (e.g., ELISA orWestern blot) can be applied to lysates of cells, and others (e.g.,immunohistological methods or fluorescence flow cytometry) can beapplied to histological sections or unlysed cell suspensions. The tissuesample can be, for example, lung, epithelial, connective, vascular,muscle, neural, skeletal, lymphatic, prostate, cervical, breast, spleen,gastric, intestinal, oral, esophageal, dermal, liver, kidney, bladder,thyroid, adrenal, brain, gallbladder, pancreatic, uterine, ovarian, ortesticular tissue.

Methods of detecting an mRNA in a tissue sample are known in the art.For example, cells can be lysed and an mRNA in the lysates or in RNApurified or semi-purified from the lysates can be detected by any of avariety of methods including, without limitation, hybridization assaysusing detectably labeled gene-specific DNA or RNA probes (e.g., NorthernBlot assays) and quantitative or semi-quantitative RT-PCR methodologiesusing appropriate gene-specific oligonucleotide primers. Alternatively,quantitative or semi-quantitative in situ hybridization assays can becarried out using, for example, tissue sections or unlysed cellsuspensions, and detectably (e.g., fluorescently or enzyme) labeled DNAor RNA probes. Additional methods for quantifying mRNA include RNAprotection assay (RPA) and SAGE.

Methods of assessing the level of B7-H1 expression (RNA and/orpolypeptide) can be can be quantitative, semi-quantitative, orqualitative. Thus, for example, the level of B7-H1 expression can bedetermined as a discrete value. For example, where quantitative RT-PCRis used, the level of expression of B7-H1 mRNA can be measured as anumerical value by correlating the detection signal derived from thequantitative assay to the detection signal of a known concentration of:(a) B7-H1 nucleic acid sequence (e.g., B7-H1 cDNA or B7-H1 transcript);or (b) a mixture of RNA or DNA that contains a nucleic acid sequenceencoding B7-H1. Alternatively, the level of B7-H1 expression can beassessed using any of a variety of semi-quantitative/qualitative systemsknown in the art. Thus, the level of expression of B7-H1 in a cell ortissue sample can be expressed as, for example, (a) one or more of“excellent”, “good”, “satisfactory”, “unsatisfactory”, and/or “poor”;(b) one or more of “very high”, “high”, “average”, “low”, and/or “verylow”; or (c) one or more of “++++”, “+++”, “++”, “+”, “+/−”, and/or “−”.Where it is desired, the level of expression of B7-H1 in tissue from asubject can be expressed relative to the expression of B7-H1 from (a) atissue of a subject known not be cancerous (e.g., a contralateral kidneyor lung, or an uninvolved lymph node); or (b) a corresponding tissuefrom one or more other subjects known not to have the cancer ofinterest, preferably known not to have any cancer.

Methods of assessing the amount of label depend on the nature of thelabel and are well known in the art. Appropriate labels include, withoutlimitation, radionuclides (e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H, or ³²P), enzymes(e.g., alkaline phosphatase, horseradish peroxidase, luciferase, orβ-glactosidase), fluorescent moieties or proteins (e.g., fluorescein,rhodamine, phycoerythrin, green fluorescent protein (GFP), or bluefluorescent protein (BFP)), or luminescent moieties (e.g., Qdot™nanoparticles supplied by the Quantum Dot Corporation, Palo Alto,Calif.). Other applicable assays include quantitativeimmunoprecipitation or complement fixation assays.

In the diagnostic assays of the invention, a subject is diagnosed ashaving cancer if the proportion of test cells from the subject thatexpress B7-H1 is greater than a control value. The control value, canbe, for example: (a) the proportion of B7-H1-expressing cells incorresponding tissue of the subject known not be cancerous (e.g., acontralateral kidney or lung, or an uninvolved lymph node); or (b) theproportion of B7-H1 expressing cells in a corresponding tissue from oneor more other subjects known not to have the cancer of interest,preferably known not to have any cancer.

The method of the invention can be used on its own or in conjunctionwith other procedures to diagnose cancer. For example, where it isdesired or preferred, the level of B7-H1-expressing test cells in atissue sample that is, or is suspected of being, cancerous can beassessed before, during, or after assessing the levels of othermolecules that are useful diagnostic cancer markers. Such diagnosticmarkers can be, without limitation, tumor-associated antigens (TAA).Relevant TAA include, without limitation, carcinoembryonic antigen(CEA), MAGE (melanoma antigen) 1-4, 6, and 12, MUC (mucin) (e.g., MUC-1,MUC-2, etc.), tyrosinase, MART (melanoma antigen), Pmel 17 (gp 100),GnT-V intron sequence (N-acetylglucosaminyltransferase V intron Vsequence), PSA (prostate-specific antigen), PSMA (prostate-specificmembrane antigen), PRAME (melanoma antigen), β-catenin, MUM-1-B(melanoma ubiquitous mutated gene product), GAGE (melanoma antigen) 1,BAGE (melanoma antigen) 2-10, c-ERB2 (HER2/neu), EBNA (Epstein-BarrVirus nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7,p53m lung resistance protein (LRP), Bcl-2, Ki-67, and VHL (vonHippel-Lindau) gene.

Method of Identifying Cancer Subjects Likely to Benefit fromImmunotherapy

Another aspect of the invention is a method of identifying a candidatefor immunotherapy. This method involves providing a tissue sample from asubject with cancer of the tissue. The tissue sample contains testcells, the test cells being cancer cells or tumor-infiltratingleukocytes. The level of test cells in the tissue sample that expressB7-H1 is assessed, such that if B7-H1 expression is not detected in thetest cells, or less than an immuno-inhibitory threshold level of thetest cells express B7-H1, the subject is more likely to benefit fromimmunotherapy.

The immuno-inhibitory threshold level is a predetermined level of therelevant test cells expressing B7-H1. If the test cells from a cancersubject of interest contain a level of B7-H1-expressing cells that isless than the immuno-inhibitory threshold level of B7-H1-expressingcells (as predetermined for the relevant cancer), that subject is morelikely to benefit from immunotherapy than another subject with the samecancer but whose corresponding test cells contain a level ofB7-H1-expressing cells equal to, or greater, than the immuno-inhibitorythreshold level. The immuno-inhibitory threshold level can be obtainedby performing statistical clinical analyses known in the art, e.g.,those described herein.

Methods of assessing whether test cells express B7-H1 are the same asthose described above for methods of diagnosis. Such methods, also asdescribed above, can be qualitative, semi-quantitative, or qualitative.

“Immunotherapy” can be active immunotherapy or passive immunotherapy.For active immunotherapy, treatment relies on the in vivo stimulation ofthe endogenous host immune system to react against tumors with theadministration of immune response-modifying agents. Theseimmune-response-modifying agents are described below.

For passive immunotherapy, treatment involves the delivery of agentswith established tumor-immune reactivity (such as immune effector cellsor antibodies) that can directly, or indirectly mediate, anti-tumoreffects and do not necessarily depend on an intact host immune system.Examples of immune effector cells include leukocytes, e.g.,tumor-infiltrating leukocytes as discussed above, T lymphocytes (such asCD8⁺ cytotoxic T lymphocytes and/or CD4⁺ T-helper lymphocytes), killercells (such as natural killer cells and lymphokine-activated killercells), B cells and antigen-presenting cells (such as dendritic cellsand macrophages).

Immunotherapy can also be one or more of the methods described below (in“Methods of Treatment” and “Methods of Inhibiting Expression of B7-H1).

Method of Prognosis

In another embodiment, the invention features a method of determiningthe prognosis of a subject with cancer. This method involves: (a)providing a tissue sample from a subject with cancer of the tissue, thetissue sample containing test cells, the test cells being cancer cellsor tumor infiltrating leukocytes; and (b) assessing the level of testcells in the tissue sample that expresses B7-H1. If a prognostic level,or more than a prognostic level, of the test cells express B7-H1, thesubject is more likely to die of the cancer than if less than aprognostic level of the test cells express B7-H1. The prognostic levelis a predetermined value obtained by performing statistical clinicalanalyses known in the art, e.g., those described herein.

Thus, for example, if test cells from a cancer subject contain asignificant level of B7-H1 expressing cells, but less than a prognosticlevel of B7-H1-expressing cells (as predetermined for the relevantcancer), the cancer subject will be no more likely to die of the cancerthan a subject with the same cancer but whose corresponding test cellscontain no detectable B7-H1-expressing cells. On the other hand, if testcells from a cancer subject contain more than a prognostic level ofB7-H1-expressing cells, the cancer subject will be more likely to die ofthe cancer than a subject with the same cancer but whose correspondingtest cells contain either no detectable B7-H1-expressing cells or alevel of B7-H1-expressing cells lower than a prognostic level ofB7-H1-expressing cells. Moreover, for subjects with cancer having levelsof B7-H1-expressing cells in appropriate test cell populations greaterthan prognostic levels, the chances of dying from the cancer is likelyto be proportional to the level of B7-H1-expressing cells in the testcell population.

As used herein, “assessing whether test cells express B7-H1” or“assessing the level of test cells in the tissue sample that expressB7-H1” can be determined by any of the methods described above. Methodsof prognosis will generally be quantitative or semi-quantitative.

Subjects can be any of those listed for “Methods of Diagnosis” andcancers can be any of the following: renal cancer, hematological cancer(e.g., leukemia or lymphoma), neurological cancer, melanoma, breastcancer, lung cancer, head and neck cancer, gastrointestinal cancer,liver cancer, pancreatic cancer, pancreatic cancer, genitourinarycancer, bone cancer, or vascular cancer

Methods of Treatment

The invention also includes a method of treatment. The method caninvolve: (a) identifying a subject with cancer, some or all cells of thecancer or some or all tumor-infiltrating leukocytes of the cancerexpressing B7-H1; and (b) delivering to the subject an agent thatinterferes with an interaction between B7-H1 and a receptor for B7-H1.These methods can be performed subsequent to, or without, performing anyof the above-described methods. The agent can be an antibody or anantibody fragment, such as, e.g., a Fab′, a F(ab′)₂, or a scFv fragmentthat binds B7-H1. The agent can also be a soluble B7-H1 or a solublefunctional fragment of B7-H1; a soluble receptor for B7-H1 or a solublefunctional fragment thereof; an antibody, or an antibody fragment, thatbinds to a receptor for B7-H1, e.g., the PD-1 receptor. The PD-1receptor is described in greater detail in U.S. Pat. No. 6,808,710, thedisclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the agent itself is administered to a subject.Generally, the agent will be suspended in a pharmaceutically-acceptablecarrier (e.g., physiological saline) and administered orally or byintravenous (i.v.) infusion, or injected subcutaneously,intramuscularly, intrathecally, intraperitoneally, intrarectally,intravaginally, intranasally, intragastrically, intratracheally, orintrapulmonarily. The agent can, for example, be delivered directly to asite of an immune response. e.g., a lymph node in the region of anaffected tissue or organ or spleen. The dosage required depends on thechoice of the route of administration; the nature of the formulation;the nature of the patient's illness; the subject's size, weight, surfacearea, age, and sex; other drugs being administered; and the judgment ofthe attending physician. Suitable dosages are in the range of0.0001-100.0 mg/kg. Wide variations in the needed dosage are to beexpected in view of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by i.v. injection. Variations in these dosage levels canbe adjusted using standard empirical routines for optimization as iswell understood in the art. Administrations can be single or multiple(e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold).Encapsulation of the compound in a suitable delivery vehicle (e.g.,polymeric microparticles or implantable devices) may increase theefficiency of delivery, particularly for oral delivery.

Alternatively, where the agent is a polypeptide, a polynucleotidecontaining a nucleic acid sequence encoding the polypeptide can bedelivered to appropriate cells in a mammal. Expression of the codingsequence can be directed to any cell in the body of the subject.However, expression will preferably be directed to cells in, or closeto, lymphoid tissue draining an affected tissue or organ. Expression ofthe coding sequence can be directed, for example, to cells comprisingthe cancer tissue (e.g., tumor-infiltrating leukocytes and tumor cells)or immune-related cells, e.g., B cells, macrophages/monocytes, orinterdigitating dendritic cells. This can be achieved by, for example,the use of polymeric, biodegradable microparticle or microcapsuledelivery devices known in the art and/or tissue or cell-specificantibodies.

Another way to achieve uptake of the nucleic acid is using liposomes,which can be prepared by standard methods. The vectors can beincorporated alone into these delivery vehicles or co-incorporated withtissue-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells [Cristiano etal. (1995), J. Mol. Med. 73:479]. Alternatively, tissue specifictargeting can be achieved by the use of tissue-specific transcriptionalregulatory elements (TRE) which are known in the art. Delivery of “nakedDNA” (i.e., without a delivery vehicle) to an intramuscular,intradermal, or subcutaneous site is another means to achieve in vivoexpression.

In the relevant polynucleotides (e.g., expression vectors), the nucleicacid sequence encoding the polypeptide of interest with an initiatormethionine and optionally a targeting sequence is operatively linked toa promoter or enhancer-promoter combination. Short amino acid sequencescan act as signals to direct proteins to specific intracellularcompartments. Such signal sequences are described in detail in U.S. Pat.No. 5,827,516, the disclosure of which is incorporated herein byreference in its entirety.

Enhancers provide expression specificity in terms of time, location, andlevel. Unlike a promoter, an enhancer can function when located atvariable distances from the transcription initiation site, provided apromoter is present. An enhancer can also be located downstream of thetranscription initiation site. To bring a coding sequence under thecontrol of a promoter, it is necessary to position the translationinitiation site of the translational reading frame of the peptide orpolypeptide between one and about fifty nucleotides downstream (3′) ofthe promoter. The coding sequence of the expression vector isoperatively linked to a transcription terminating region.

Suitable expression vectors include plasmids and viral vectors such asherpes viruses, retroviruses, vaccinia viruses, attenuated vacciniaviruses, canary pox viruses, adenoviruses and adeno-associated viruses,among others.

Polynucleotides can be administered in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are biologicallycompatible vehicles that are suitable for administration to a human,e.g., physiological saline or liposomes. A therapeutically effectiveamount is an amount of the polynucleotide that is capable of producing amedically desirable result (e.g., decreased proliferation of cancercells) in a treated subject. As is well known in the medical arts, thedosage for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Dosages will vary, but apreferred dosage for administration of polynucleotide is fromapproximately 10⁶ to approximately 10¹² copies of the polynucleotidemolecule. This dose can be repeatedly administered, as needed. Routes ofadministration can be any of those listed above.

In addition, the method can be an ex vivo procedure that involvesproviding a recombinant cell which is, or is a progeny of a cell,obtained from a subject and has been transfected or transformed ex vivowith one or more nucleic acids encoding one or more agents thatinterfere with an interaction between B7-H1 and a receptor for B7-H1, sothat the cell expresses the agent(s); and administering the cell to thesubject. The cells can be cells obtained from a cancer tissue (e.g.,tumor cells and/or tumor-infiltrating leukocytes) or from anon-cancerous tissue obtained preferably from a subject to whom thesecells are to be administered or from another subject. The donor andrecipient of the cells can have identical major histocompatibilitycomplex (MHC; HLA in humans) haplotypes. Optimally, the donor andrecipient are homozygotic twins or are the same individual (i.e., areautologous). The recombinant cells can also be administered torecipients that have no, or only one, two, three, or four MHC moleculesin common with the recombinant cells, e.g., in situations where therecipient is severely immunocompromised, where only mismatched cells areavailable, and/or where only short term survival of the recombinantcells is required or desirable.

The efficacy of the agent can be evaluated both in vitro and in vivo.Briefly, the agent can be tested for its ability, for example, to (a)inhibit the interaction between B7-H1 and a receptor for B7-H1, (b)inhibit growth of cancer cells, (c) induce death of cancer cells, or (d)render the cancer cells more susceptible to cell-mediated immuneresponses generated by leukocytes (e.g., lymphocytes and/ormacrophages). For in vivo studies, the agent can, for example, beinjected into an animal (e.g., a mouse cancer model) and its effects oncancer are then assessed. Based on the results, an appropriate dosagerange and administration route can be determined.

As used throughout the present application, the term “antibody” refersto a whole antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecule that isgenerated by any one of a variety of methods that are known in the art.The antibody can be a polyclonal or a monoclonal antibody. Also usefulfor the invention are chimeric antibodies and humanized antibodies madefrom non-human (e.g., mouse, rat, gerbil, or hamster) antibodies. Asused herein, the term “antibody fragment” refers to an antigen-bindingfragment, e.g., Fab, F(ab′)₂, Fv, and single chain Fv (scFv) fragments.An scFv fragment is a single polypeptide chain that includes both theheavy and light chain variable regions of the antibody from which thescFv is derived.

Antibody fragments that contain the binding domain of the molecule canbe generated by known techniques. For example: F(ab′)₂ fragments can beproduced by pepsin digestion of antibody molecules; and Fab fragmentscan be generated by reducing the disulfide bridges of F(ab′)₂ fragmentsor by treating antibody molecules with papain and a reducing agent. See,e.g., National Institutes of Health, 1 Current Protocols In Immunology,Coligan et al., ed. 2.8, 2.10 (Wiley Interscience, 1991). scFv fragmentscan be produced, for example, as described in U.S. Pat. No. 4,642,334,which is incorporated herein by reference in its entirety.

Chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example, using methodsdescribed in Robinson et al., International Patent PublicationPCT/US86/02269; Akira et al., European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., PCT ApplicationWO 86/01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al. (1988) Science 240,1041-43; Liu et al. (1987) J. Immunol. 139, 3521-26; Sun et al. (1987)PNAS 84, 214-18; Nishimura et al. (1987) Canc. Res. 47, 999-1005; Woodet al. (1985) Nature 314, 446-49; Shaw et al. (1988) J. Natl. CancerInst. 80, 1553-59; Morrison, (1985) Science 229, 1202-07; Oi et al.(1986) BioTechniques 4, 214; Winter, U.S. Pat. No. 5,225,539; Jones etal. (1986) Nature 321, 552-25; Veroeyan et al. (1988) Science 239, 1534;and Beidler et al. (1988) J. Immunol. 141, 4053-60.

As used herein, a “functional fragment” of a B7-H1 receptor means afragment of a receptor for B7-H1 that is smaller than the wild-typemature B7-H1 receptor and has at least 10% (e.g., at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 98%, at least99%, or 100% or more) of the ability of the wild-type mature receptorfor B7-H1 to bind to B7-H1. As used herein, a “functional fragment” ofB7-H1 means a fragment of the wild-type mature B7-H1 polypeptide that issmaller than the wild-type mature B7-H1 polypeptide and has at least 10%(e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, at least 99%, or 100% or more) of the ability of thewild-type mature B7-H1 to bind to the B7-H1 receptor. Methods of testingand comparing the ability of molecules to bind to each other are knownto those in the art.

As used herein, the term “soluble” distinguishes the receptors used inthe present invention from their cell membrane-bound counterparts. Asoluble receptor, or a soluble functional fragment of a receptor cancontain, for example, an extracellular (ligand binding) domain, but lackthe transmembrane region that causes retention of a receptor on the cellsurface. Methods of producing soluble receptors or fragments thereof areknown in the art and include, for example, expressing a DNA fragmentencoding an extracellular domain of a receptor in a suitable hostcell/expression vector system.

The term “treatment”, as used herein, means administration of an agentto a subject, who has cancer (or is suspected of having cancer), withthe purpose to cure, alleviate, relieve, remedy, prevent, or amelioratethe disorder, the symptom of the disorder, the disease state secondaryto the disorder, or the predisposition toward the disorder. An“effective amount” of a therapeutic agent (or composition) is an amountof the agent (or composition) that is capable of producing a medicallydesirable result in a treated subject. The method of the invention canbe performed alone or in conjunction with other drugs or therapy.

As used herein, “prophylaxis” can mean complete prevention of thesymptoms of a disease, a delay in onset of the symptoms of a disease, ora lessening in the severity of subsequently developed disease symptoms.As used herein, “therapy” can mean a complete abolishment of thesymptoms of a disease or a decrease in the severity of the symptoms ofthe disease.

Methods of Inhibiting Expression of B7-H1

Another aspect of the invention is a method of inhibiting the expressionof B7-H1 in a tumor cell or a tumor-infiltrating leukocyte. The methodinvolves: (a) identifying a subject with cancer, the cancer containing atarget cell that expresses B7-H1, the target cell being a tumor cell ora tumor-infiltrating leukocyte; and (b) introducing into the targetcell: (i) an antisense oligonucleotide that hybridizes to a B7-H1transcript, the antisense oligonucleotide inhibiting the expression ofB7-H1 in the cell; or (ii) a B7-H1 interference RNA (RNAi). Thesemethods can be performed subsequent to, or without, performing any ofthe above-described methods.

Since, as noted above, aberrant B7-H1 expression impairs the functionand survival of tumor-specific T cells, it is likely that by inhibitingthe cellular expression of B7-H1, as well as by interfering with theinteraction between B7-H1 and its receptor, the anti-tumor immuneresponses can be restored. Thus, the method can be useful for therapyand/or prophylaxis of any cancer recited herein. The method can be used,for example, in the treatment of RCC.

Antisense compounds are generally used to interfere with proteinexpression either by, for example, interfering directly with translationof a target mRNA molecule, by RNAse-H-mediated degradation of the targetmRNA, by interference with 5′ capping of mRNA, by prevention oftranslation factor binding to the target mRNA by masking of the 5′ cap,or by inhibiting of mRNA polyadenylation. The interference with proteinexpression arises from the hybridization of the antisense compound withits target mRNA. A specific targeting site on a target mRNA of interestfor interaction with an antisense compound is chosen. Thus, for example,for modulation of polyadenylation, a preferred target site on an mRNAtarget is a polyadenylation signal or a polyadenylation site. Fordiminishing mRNA stability or degradation, destabilizing sequences arepreferred target sites. Once one or more target sites have beenidentified, oligonucleotides are chosen which are sufficientlycomplementary to the target site (i.e., hybridize sufficiently wellunder physiological conditions and with sufficient specificity) to givethe desired effect.

With respect to this invention, the term “oligonucleotide” refers to anoligomer or polymer of RNA, DNA, or a mimetic of either. The termincludes oligonucleotides composed of naturally-occurring nucleobases,sugars, and covalent internucleoside (backbone) linkages. The normallinkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester bond.The term also refers however to oligonucleotides composed entirely of,or having portions containing, non-naturally occurring components whichfunction in a similar manner to the oligonucleotides containing onlynaturally-occurring components. Such modified substitutedoligonucleotides are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for target sequence, and increased stability in thepresence of nucleases. In the mimetics, the core base (pyrimidine orpurine) structure is generally preserved but (1) the sugars are eithermodified or replaced with other components and/or (2) theinter-nucleobase linkages are modified. One class of nucleic acidmimetic that has proven to be very useful is referred to as proteinnucleic acid (PNA). In PNA molecules the sugar backbone is replaced withan amide-containing backbone, in particular an aminoethylglycinebackbone. The bases are retained and are bound directly to the azanitrogen atoms of the amide portion of the backbone. PNA and othermimetics useful in the instant invention are described in detail in U.S.Pat. No. 6,210,289, the disclosure of which is incorporated herein byreference in its entirety.

The antisense oligomers to be used in the methods of the inventiongenerally comprise about 8 to about 100 (e.g., about 14 to about 80 orabout 14 to about 35) nucleobases (or nucleosides where the nucleobasesare naturally occurring).

The antisense oligonucleotides can themselves be introduced into a cellor an expression vector containing a nucleic sequence (operably linkedto a TRE) encoding the antisense oligonucleotide can be introduced intothe cell. In the latter case, the oligonucleotide produced by theexpression vector is an RNA oligonucleotide and the RNA oligonucleotidewill be composed entirely of naturally occurring components.

Where antisense oligonucleotides per se are administered, they can besuspended in a pharmaceutically-acceptable carrier (e.g., physiologicalsaline) and administered under the same conditions described above foragents that interfere with an interaction between B7-H1 and a receptorfor B7-H1.

Where an expression vector containing a nucleic sequence (operablylinked to a TRE) encoding the antisense oligonucleotide is administeredto a subject, expression of the coding sequence can be directed to atumor cell of tumor-infiltrating leukocyte in the body of the subjectusing any of the cell- or tissue-targeting techniques described abovefor vectors that express polypeptides that interfere with an interactionbetween B7-H1 and a receptor for B7-H1.

Double-stranded interfering RNA (RNAi) homologous to B7-H1 DNA can alsobe used to reduce expression of B7-H1 in tumor cells and/ortumor-infiltrating leukocytes. See, e.g., Fire et al. (1998) Nature391:806-811; Romano and Masino (1992) Mol. Microbiol. 6:3343-3353;Cogoni et al. (1996) EMBO J. 15:3153-3163; Cogoni and Masino (1999)Nature 399:166-169; Misquitta and Paterson (1999) Proc. Natl. Acad. Sci.USA 96:1451-1456; and Kennerdell and Carthew (1998) Cell 95:1017-1026.The disclosures of all these articles are incorporated herein byreference in their entirety.

The sense and anti-sense RNA strands of RNAi can be individuallyconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, each strand can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecule or to increase the physical stability of theduplex formed between the sense and anti-sense strands, e.g.,phosphorothioate derivatives and acridine substituted nucleotides. Thesense or anti-sense strand can also be produced biologically using anexpression vector into which a target B7-H1 sequence (full-length or afragment) has been subcloned in a sense or anti-sense orientation. Thesense and anti-sense RNA strands can be annealed in vitro beforedelivery of the dsRNA to cells. Alternatively, annealing can occur invivo after the sense and anti-sense strands are sequentially deliveredto tumor cells and/or tumor-infiltrating leukocytes.

Double-stranded RNAi interference can also be achieved by introducinginto tumor cells and/or tumor-infiltrating leukocytes a polynucleotidefrom which sense and anti-sense RNAs can be transcribed under thedirection of separate promoters, or a single RNA molecule containingboth sense and anti-sense sequences can be transcribed under thedirection of a single promoter.

It will be understood that certain drugs and small molecules can also beused inhibit expression of B7-H1 in tumor cells and/ortumor-infiltrating leukocytes.

One of skill in the art will appreciate that RNAi, drug, and smallmolecule methods can be, as for the antisense methods described above,in vitro and in vivo. Moreover, methods and conditions of delivery arethe same as those for antisense oligonucleotides.

In any of the above methods of inhibiting the interaction between B7-H1and a receptor for B7-H1 and of inhibiting expression of B7-H1, one ormore agents (e.g., two, three, four, five, six, seven, eight, nine, ten,11, 12, 15, 18, 20, 25, 30, 40, 50, 60, 70, 80, 100, or more) including,for example, inhibitory compounds, antisense oligonucleotides, RNAi,drugs, or small molecules (or vectors encoding them), can be used.

Moreover, such agents can be used together with one or more (e.g., two,three, four, five, six, seven, eight, nine, ten, 11, 12, 15, 18, 20, 25,30, 40, 50, 60, 70, 80, 100, or more) supplementary agents, includingimmunomodulatory cytokines, growth factors, antiangiogenic factors,immunogenic stimuli, and/or antibodies specific for any of these. Suchsupplementary agents can administered before, simultaneous with, orafter delivery of any of the above-listed agents.

Examples of immunomodulatory cytokines, growth factors, andantiangiogenic factors include, without limitation, interleukin (IL)-1to 25 (e.g., IL-2, IL-12, or IL-15), interferon-γ (IFN-γ), interferon-α(IFN-α), interferon-β (IFN-β), tumor necrosis factor-α (TNF-α),granulocyte macrophage colony stimulating factor (GM-CSF), granulocytemacrophage colony stimulating factor (G-CSF), endostatin, angiostatin,and thrombospondin. Immunomodulatory cytokines, growth factors,antiangiogenic factors include substances that serve, for example, toinhibit infection (e.g., standard anti-microbial antibiotics), inhibitactivation of T cells, or inhibit the consequences of T cell activation.For example, where it is desired to decrease a Th1-type immune response(e.g., in a DTH response), a cytokine such as interleukin (IL)-4, IL-10,or IL-13 or an antibody specific for a cytokine such as IL-12 orinterferon-γ (IFN-γ) can be used. Alternatively, where it is desired toinhibit a Th2-type immune response (e.g., in an immediate typehypersensitivity response), a cytokine such as IL-12 or IFN-γ or anantibody specific for IL-4, IL-10, or IL-13 can be used as asupplementary agent. Also of interest are antibodies (or any of theabove-described antibody fragments or derivatives) specific forproinflammatory cytokines and chemokines such as IL-1, IL-6, IL-8, tumornecrosis factor-α (TNF-α), macrophage inflammatory protein (MIP)-1,MIP-3α, monocyte chemoattractant protein-1 (MCP-1), epithelialneutrophil activating peptide-78 (ENA-78), interferon-γ-inducibleprotein-10 (IP10), Rantes, and any other appropriate cytokine orchemokine recited herein.

In some instances, it may be desired to increase the immune response ina subject by the administration of one or more immune responsemodifying-agents. Such immune response-modifying agents include, inaddition to any of the immunomodulatory cytokines, growth factors, andangiogenic factors listed above, immunogenic stimuli that can bedelivered via the antigen-specific T cell receptor (TCR) expressed onthe surface of the T cell. More commonly, but not necessarily, such astimulus is provided in the form of an antigen for which the TCR isspecific. While such antigens will generally be protein, they can alsobe carbohydrates, lipids, nucleic acids or hybrid molecules havingcomponents of two or more of these molecule types, e.g., glycoproteinsor lipoproteins. However, the immunogenic stimulus can also be providedby other agonistic TCR ligands such as antibodies specific for TCRcomponents (e.g., TCR α-chain or β-chain variable regions) or antibodiesspecific for the TCR-associated CD3 complex. Antigens useful asimmunogenic stimuli include alloantigens (e.g., a MHC alloantigen) on,for example, an antigen presenting cell (APC) (e.g., a dendritic cell(DC), a macrophage, a monocyte, or a B cell). DC of interest areinterdigitating DC and not follicular DC; follicular DC present antigento B cells. For convenience, interdigitating DC are referred to hereinas DC. Methods of isolating DC from tissues such as blood, bone marrow,spleen, or lymph node are known in the art, as are methods of generatingthem in vitro from precursor cells in such tissues.

Also useful as immunogenic stimuli are polypeptide antigens andpeptide-epitopes derived from them (see below). Unprocessed polypeptidesare processed by APC into peptide-epitopes that are presented toresponsive T cells in the form of molecular complexes with MHC moleculeson the surface of the APC. Useful immunogenic stimuli also include asource of antigen such as a lysate of either tumor cells or cellsinfected with an infectious microorganism of interest. APC (e.g., DC)pre-exposed (e.g., by coculturing) to antigenic polypeptides,peptide-epitopes of such polypeptides or lysates of tumor (or infectedcells) can also be used as immunogenic stimuli. Such APC can also be“primed” with antigen by culture with a cancer cell or infected cell ofinterest; the cancer or infected cells can optionally be irradiated orheated (e.g., boiled) prior to the priming culture. In addition, APC(especially DC) can be “primed” with either total RNA, mRNA, or isolatedTAA-encoding RNA.

Alternatively, an immunogenic stimulus be provided in the form of cells(e.g., tumor cells or infected cells producing the antigen of interest).In addition, immunogenic stimuli can be provided in the form of cellhybrids formed by fusing APC (e.g., DC) with tumor cells [Gong et al.(2000) Proc. Natl. Acad. Sci. USA 97(6):2716-2718; Gong et al. (1997)Nature Medicine 3(5):558-561; Gong et al. (2000) J. Immunol.165(3):1705-1711] or infected cells of interest.

Also useful as immunogenic stimuli are heat shock proteins bound toantigenic peptide-epitopes derived from antigens (e.g., tumor-associatedantigens or antigens produced by infectious microorganisms) [Srivastava(2000) Nature Immunology 1(5):363-366]. Heat shock proteins of interestinclude, without limitation, glycoprotein 96 (gp96), heat shock protein(hsp) 90, hsp70, hsp110, glucose-regulated protein 170 (grp170) andcalreticulin. Immunogenic stimuli can include one or more (e.g., one,two, three, four, five, six, seven, eight, nine, ten, more) heat shockproteins isolated from tumor cells. Such tumor are preferably, but notnecessarily, from the same subject (i) to whom the agent that interfereswith the interaction between B7-H1 and a receptor for B7-H1 is to bedelivered or (ii) in whose tumor cells or tumor infiltrating leukocytesthe expression of B7-H1 is to be inhibited. The tumor cells can also beobtained, for example, from another individual having the same as thesubject, or a related tumor-type. Alternatively, the heat shock proteincan be isolated from mammalian cells expressing a transcriptosomeprepared from tumor cells of interest.

As indicated above, immunogenic stimuli useful in the invention can beany of a wide variety of tumor cells, APC “primed” with tumor cells,hybrid cells, or TAA (see above), peptide-epitopes of such TAA, and APC“primed” with TAA or peptide-epitopes of them. As used herein, a “TAA”is a molecule (e.g., a protein molecule) that is expressed by a tumorcell and either (a) differs qualitatively from its counterpart expressedin normal cells, or (b) is expressed at a higher level in tumor cellsthan in normal cells. Thus, a TAA can differ (e.g., by one or more aminoacid residues where the molecule is a protein) from, or it can beidentical to, its counterpart expressed in normal cells. It ispreferably not expressed by normal cells. Alternatively, it is expressedat a level at least two-fold higher (e.g., a two-fold, three-fold,five-fold, ten-fold, 20-fold, 40-fold, 100-fold, 500-fold, 1,000-fold,5,000-fold, or 15,000-fold higher) in a tumor cell than in the tumorcell's normal counterpart. Relevant TAA include, without limitation, anyof the TAAs listed above.

Administrations of the agents and/or the one or more supplementaryagents can be systemic (e.g., intravenous) or local, e.g., duringsurgery by direct injection or infusion into the tissue that comprisesthe cells of the cancer and/or tumor-infiltrating leukocytes. Theadministrations can also be by any of routes, doses, and schedulesrecited herein.

In addition, it is understood that the above-described methods can beused in combination with any one of a variety of other therapeuticmodalities known in the art, such as, without limitation, chemotherapy,immunotherapy, radiotherapy, or gene therapy.

In both of the methods of inhibiting the interaction between B7-H1 and areceptor for B7-H1 and the methods of inhibiting expression of B7-H1,the cancer can be any cancer recited herein and includes, e.g., renalcell carcinoma. Subjects can be mammals and include, for example,humans, non-human primates (e.g., monkeys, baboons, or chimpanzees),horses, cows (or oxen or bulls), pigs, sheep, goats, cats, rabbits,guinea pigs, hamsters, rats, gerbils, or mice.

The following examples are meant to illustrate, not limit, theinvention.

Examples Example 1 Materials and Methods Patient Selection

Upon approval from the Mayo Clinic Institutional Review Board, 429patients were identified from Mayo Clinic Nephrectomy Registry that werepreviously treated with radical nephrectomy or nephron-sparing surgeryfor unilateral, sporadic clear cell RCC between 2000 and 2002. Sincepathologic features and patient outcome differ by RCC subtype, allanalyses were restricted to patients treated with clear cell RCC only,the most common of the RCC subtypes [Cheville et al. (2003) Am. J. Surg.Pathol. 27:612-624]. Since the hB7-H1-specific monoclonal antibody, 5H1(see below), can reproducibly stain fresh-frozen, but notparaffin-fixed, tissue [Dong et al. (2002) Nature Med. 8:793-800],patients were selected based on availability of fresh-frozen tissue.

Pathologic Features

The pathologic features examined included histologic subtype, tumorsize, primary tumor stage, regional lymph node involvement, and distantmetastases at nephrectomy (2002 TNM), nuclear grade, and histologictumor necrosis. The microscopic slides from all specimens were reviewedby a urologic pathologist without prior knowledge of patient outcome.Histologic subtype was classified according to the Union InternationaleContre le Cancer, American Joint Committee on Cancer, and Heidelbergguidelines [Storkel et al. (1997) Cancer 80:987-989; Kovacs et al.(1997) J. Pathol. 183:131-133]. Nuclear grade was assigned usingstandardized criteria [Lohse et al. (2002) Am. J. Clin. Pathol.118:877-886]. Histologic tumor necrosis was defined as the presence ofany microscopic coagulative tumor necrosis. Degenerative changes such ashyalinization, hemorrhage, and fibrosis were not considered necrosis.

Immunohistochemical Staining of Tumor Specimens

Cryosections generated from RCC tumors and normal renal corticalspecimens (5 μm thickness) were mounted on Superfrost Plus slides, airdried, and fixed in ice-cold acetone. Sections were stained using theDako Autostainer and Dako Cytomation Labeled Polymer (EnVision+) HRPdetection Kit™ (Dako; Carpinteria, Calif.). Slides were blocked withH₂O₂ for 10 minutes followed by incubation with the primary anti-B7-H1antibody for 30 minutes at room temperature. A horseradishperoxidase-conjugated secondary reagent (goat anti-mouse immunoglobulin)was then applied to the slides at room temperature for 15 minutesfollowed by incubation with chromogen-substrate for 10 minutes. Finally,sections were counter-stained for 3 minutes with modified Schmidt'sHematoxylin. The primary antibody used in this study was 5H1, a mouseanti-hB7-H1 monoclonal antibody [Dong et al. (2002) Nature Med.8:793-800). Benign renal tumors and peripheral T cells were not stainedin this study. Positive tissues controls for hB7-H1 staining were humantonsillar tissues. Irrelevant isotype-matched antibodies were used tocontrol for non-specific staining.

Quantification of hB7-H1 Expression

The percentages of tumor cells and leukocytes that stained positive forhB7-H1 were quantified in 5-10% increments by a urologic pathologistwithout prior knowledge of patient outcome. The extent of leukocyticinfiltration was assessed and recorded as absent, focal (scatteredlymphoid aggregates), moderate, or marked. An adjusted scorerepresenting leukocytic hB7-H1 expression was calculated as thepercentage of leukocytes that stained positive for hB7-H1 multiplied bythe extent of leukocytic infiltration (0=absent, 1=focal, 2=moderate,3=marked).

Statistical Methods

Comparisons between the pathologic features and hB7-H1 expression wereevaluated using chi-square, Fisher's exact and Wilcoxon rank sum tests.Cancer-specific survival was estimated using the Kaplan-Meier method.The duration of follow-up was calculated from the date of nephrectomy tothe date of death or last follow-up. Cause of death was determined fromthe death certificate or physician correspondence. Scatter plots of thepercentage of cells that stained positive for hB7-H1 versus thedifference in observed survival and the survival expected from a Coxproportional hazards regression model (formally known as a Martingaleresidual) were used to identify potential cut-off points for hB7-H1expression [Therneau et al. (2000) Modeling Survival Data: Extending theCox Model, ed. 1 (Springer-Verlag, Ann Arbor), pp. 87-92]. Theassociations of these cut points with death from RCC were evaluatedusing Cox proportional hazards regression models univariately and afteradjusting for primary tumor stage, regional lymph node involvement,distant metastases, tumor size, nuclear grade, and histologic tumornecrosis, one feature at a time. The association of hB7-H1 expressionwith death from RCC was also adjusted for the Mayo Clinic SSIGN (Stage,Size, Grade, and Necrosis) Score, a prognostic composite scorespecifically developed for patients with clear cell RCC [Frank et al.(2002) J. Urol. 168:2395-2400]. Statistical analyses were performedusing the SAS software package (SAS Institute, Cary, N.C.) and p-values<0.05 were considered statistically significant.

Example 2 Survival of RCC Patients with Fresh-Frozen Tissue SamplesAvailable

Of the 429 patients eligible for the study, 196 (46%) had fresh-frozentissue available for laboratory investigation. Patients withfresh-frozen tissues had larger tumors compared with those who did not(median tumor size 6.0 cm versus 5.0 cm; p=0.008). However, no otherfeature studied was significantly different between the two groups.Furthermore, there was not a statistically significant difference incancer-specific survival between patients with and without fresh-frozentissues (p=0.314).

At last follow-up, 39 of the 196 patients studied had died, including 30patients who died from clear cell RCC at a median of 1.1 years followingnephrectomy (range 0-2.5). Among the 157 patients who were still aliveat last follow-up, the median duration of follow-up was 2.0 years (range0-4.1). The estimated cancer-specific survival rates (standard error,number still at risk) at 1, 2, and 3 years following nephrectomy were91.4% (2.1%, 148), 81.8% (3.3%, 78), and 77.9% (3.8%, 30), respectively.

Example 3 Correlation of hB7-H1 Expression in RCC Tumor Cells withPatient Outcome

Immunohistochemical staining of the 196 clear cell RCC specimensrevealed either no hB7-H1 expression by RCC tumor cells, or varyingdegrees of hB7-H1 expressed by either RCC tumor cells and/or RCCtumor-infiltrating leukocytes (Tables 1 and 2 and FIG. 1). In addition,proximal tubules within the renal cortex, from which RCC tumors arebelieved to arise, exhibited no hB7-H1 expression among the 20 normalrenal cortical specimens studied (FIG. 1).

The percentages of tumor cells that stained positive for hB7-H1 for the196 specimens studied are summarized in Table 1. A scatter plot of tumorhB7-H1 expression versus the expected risk of death for each patientsuggested that a cut point of 10% would be appropriate for these data.There were 73 (37.2%) patients with specimens that had ≧10% tumor cellhB7-H1 expression.

TABLE 1 Percent Tumor HB7-H1 Expression in 196 Clear Cell RCC Specimens% hB7-H1 Expression N (%) 0 66 (33.7) 5 57 (29.1) 10 27 (13.8) 15 4(2.0) 20 15 (7.7)  25 3 (1.5) 30 6 (3.1) 40 2 (1.0) 50 4 (2.0) 60 3(1.5) 70 3 (1.5) 80 2 (1.0) 90 3 (1.5) 100 1 (0.5)

TABLE 2 Adjusted Score for Leukocyte hB7-H1 Expression in 196 Clear CellRCC Specimens Leukocytic % hB7-H1 Adjusted Infiltration* ExpressionScore N (%) 0 0 0 81 (41.3) 1 5 5 4 (2.0) 1 10 10 1 (0.5) 1 30 30 2(1.0) 1 50 50 4 (2.0) 1 60 60 3 (1.5) 1 70 70 22 (11.2) 1 80 80 12(6.1)  1 90 90 10 (5.1)  2 5 10 3 (1.5) 2 10 20 4 (2.0) 2 20 40 2 (1.0)2 30 60 2 (1.0) 2 50 100 6 (3.1) 2 60 120 1 (0.5) 2 70 140 9 (4.6) 2 80160 7 (3.6) 2 90 180 8 (4.1) 3 5 15 1 (0.5) 3 20 60 1 (0.5) 3 30 90 4(2.0) 3 70 210 2 (1.0) 3 80 240 4 (2.0) 3 90 270 2 (1.0) 3 100 300 1(0.5) *The extent of leukocytic infiltration was recorded as 0 = absent,1 = focally present, 2 = moderately present, or 3 = markedly present.

The extent of leukocytic infiltration was recorded as 0=absent,1-focally present, 2=moderately present, or 3=markedly present. Theassociations of tumor hB7-H1 expression with death from RCC, bothunivariately and after adjusting for TNM stage, tumor size, nucleargrade, and histologic tumor necrosis are shown in Table 3. Univariately,patients with specimens that had ≧10% tumor hB7-H1 expression were closeto 3 times more likely to die from RCC compared with patients withspecimens that had <10% expression (risk ratio 2.91; 95% CI 1.39-6.13;p=0.005; FIG. 2A). In multivariate analyses, patients with specimensthat had ≧10% tumor hB7-H1 expression were significantly more likely todie from RCC, even after adjusting for primary tumor stage, distantmetastases, or primary tumor size.

TABLE 3 Associations of hB7-H1 Expression with Death from RCC in 196Clear Cell RCC Specimens Tumor hB7-H1 Expression ≧10% Risk Ratio (95%CI)* P-value Univariate Model 2.91 (1.39-6.13) 0.005 Adjusted for: 2002Primary Tumor Stage (T) 2.83 (1.34-5.96) 0.006 Regional Lymph Node 1.97(0.87-4.45) 0.103 Involvement (N) Distant Metastases (M) 2.24(1.06-4.73) 0.035 Primary Tumor Size 2.88 (1.37-6.06) 0.005 NuclearGrade 1.96 (0.90-4.30) 0.092 Histologic Tumor Necrosis 1.69 (0.78-3.65)0.183 Leukocytic hB7-H1 Expression ≧100 Univariate Model 3.58(1.74-7.37) <0.001 Adjusted for: 2002 Primary Tumor Stage (T) 3.34(1.62-6.90) 0.001 Regional Lymph Node 3.59 (1.74-7.41) <0.001Involvement (N) Distant Metastases (M) 2.16 (1.03-4.53) 0.042 PrimaryTumor Size 2.64 (1.27-5.46) 0.009 Nuclear Grade 3.03 (1.46-6.29) 0.003Histologic Tumor Necrosis 2.87 (1.39-5.95) 0.004 High AggregateIntratumoral hB7-H1 Expression Univariate Model  4.53 (1.94-10.56)<0.001 Adjusted for: 2002 Primary Tumor Stage (T) 4.07 (1.74-9.51) 0.001Regional Lymph Node 3.36 (1.39-8.16) 0.007 Involvement (N) DistantMetastases (M) 3.12 (1.32-7.38) 0.009 Primary Tumor Size 4.25(1.82-9.91) <0.001 Nuclear Grade 3.09 (1.28-7.50) 0.012 Histologic TumorNecrosis 2.68 (1.12-6.42) 0.027 *Risk ratios represent the risk of deathfrom clear cell RCC for the feature listed, either univariately or aftermultivariate adjustment. For example, patients with specimens that had≧10% tumor hB7-H1 expression were 2.9 times more likely to die from RCCcompared with patients with specimens that had <10% tumor hB7-H1expression, even after adjusting for primary tumor size (p = 0.005).

The adjusted scores for leukocytic hB7-H1 expression are summarized inTable 2. There were 40 (20.4%) specimens with an adjusted leukocytehB7-H1 score of 100 or greater (essentially moderate or markedleukocytic infiltration with at least 50% of the leukocytes stainingpositive for hB7-H1), which appeared to be a reasonable cut point toexamine and illustrate the association of this feature with patientoutcome. The associations of leukocyte hB7-H1 expression with death fromRCC are summarized in Table 3. Univariately, patients with specimensthat had an adjusted leukocyte hB7-H1 score ≧100 were 3.6 times morelikely to die from RCC compared with patients that had specimens withscores <100 (risk ratio 3.58; 95% CI 1.74-7.37; p<0.001; FIG. 2B).Patients with specimens that demonstrated high levels of leukocytehB7-H1 expression were significantly more likely to die from RCC evenafter adjusting for TNM stage, primary tumor size, nuclear grade, orhistologic tumor necrosis.

Since both tumor and leukocyte hB7-H1 expression were significantlyassociated with patient outcome both univariately and after multivariateadjustment, the combination of these two features were evaluated. Therewere 87 (44.4%) specimens that had either ≧10% tumor hB7-H1 expressionor an adjusted score for leukocyte hB7-H1 expression ≧100 (i.e., highaggregate intratumoral hB7-H1 expression). Twenty-six (13.3%) of thesespecimens had both features. Conversely, 109 (55.6%) specimens had <10%tumor hB7-H1 expression and <100 leukocyte hB7-H1 expression (i.e., lowaggregate intratumoral hB7-H1 expression). The associations of thiscombined feature with death from RCC are summarized in Table 3.Univariately, patients with specimens that had high aggregateintratumoral hB7-H1 expression were 4.5 times more likely to die fromRCC compared with patients with specimens that had both <10% tumorexpression and <100 leukocyte expression (risk ratio 4.53; 95% CI1.94-10.56; p<0.001). After adjusting for the Mayo Clinic SSIGN Score,patients with high aggregate intratumoral hB7-H1 expression remainedover twice as likely to die from RCC compared with patients with lowaggregate intratumoral hB7-H1, although this difference did not attainstatistical significance (risk ratio 2.19; 95% CI 0.91-5.24; p=0.079).However, patients with specimens that had high aggregate intratumoralhB7-H1 expression were significantly more likely to die from RCC afteradjusting for TNM stage, primary tumor size, nuclear grade, andhistologic tumor necrosis, one feature at a time. The association ofcombined tumor and leukocyte hB7-H1 expression with the pathologicfeatures under study were also investigated. High aggregate intratumoralhB7-H1 expression levels were significantly associated with regionallymph node involvement, distant metastases, advanced nuclear grade, andthe presence of histologic tumor necrosis (Table 4).

TABLE 4 Associations of Tumor and Leukocyte hB7-H1 Expression withPathologic Features in 196 Clear Cell RCC Specimens High AggregateIntratumoral HB7-H1 Expression No Yes N = 109 N = 87 Feature N (%)P-value 2002 Primary Tumor Stage pT1 and pT2 88 (80.7) 62 (71.3) 0.120pT3 and pT4 21 (19.3) 25 (28.7) Regional Lymph Node Involvement pNx andpN0 108 (99.1)  76 (87.4) <0.001 pN1 and pN2 1 (0.9) 11 (12.6) DistantMetastases pM0 99 (90.8) 69 (79.3) 0.022 pM1 10 (9.2)  18 (20.7) PrimaryTumor Size <5 cm 46 (42.2) 25 (28.7) 0.051 ≧5 cm 63 (57.8) 62 (71.3)Nuclear Grade 1 and 2 69 (63.3) 23 (26.4) <0.001 3 36 (33.0) 50 (57.5) 44 (3.7) 14 (16.1) Histologic Tumor Necrosis Absent 94 (86.2) 55 (63.2)<0.001 Present 15 (13.8) 32 (36.8)

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method for the immunotherapy treatment of asubject with cancer, comprising: (a) identifying whether the subject issuitable for immunotherapy, the identifying comprising providing atissue sample from the subject, which contains test cells, wherein thetest cells comprise tumor cells and/or tumor-infiltrating leukocytes,and assessing the proportion of test cells in the tissue sample thatexpress B7-H1, the identification being made if <10% of the tumor cellsexpress B7-H1 and/or if an adjusted expression score of thetumor-infiltrating leukocytes is <100; and (b) administering an agentfor immunotherapy to the subject, wherein said cancer is renal cellcarcinoma.
 2. The method of claim 1, wherein the subject is a human. 3.The method of claim 1, wherein the immunotherapy treatment is a passiveimmunotherapy treatment.
 4. The method of claim 1, wherein theimmunotherapy treatment is an active immunotherapy treatment.
 5. Themethod of claim 1, wherein said agent for immunotherapy is at least oneagent selected from the group consisting of interleukin-2,interleukin-4, interleukin-10, interleukin-12, interleukin-13,interferon-gamma, an antibody specific to interferon-gamma, an antibodyspecific to interleukin-1, an antibody specific to interleukin-4, anantibody specific to interleukin-6, an antibody specific tointerleukin-8, an antibody specific to interleukin-10, an antibodyspecific to interleukin-12, an antibody specific to interleukin-13, anantibody specific to tumor necrosis factor-alpha, an antibody specificto macrophage inflammatory protein-1, an antibody specific to macrophageinflammatory protein-3alpha, an antibody specific to monocytechemoattractant protein-1, an antibody specific to epithelial neutrophilactivating peptide-78, an antibody specific tointerferon-gamma-inducible protein-10, interferon-gamma,interferon-alpha, interferon-beta, tumor necrosis factor-alpha,granulocyte macrophage colony stimulating factor, T-cells, killer cells,B cells, and antigen-presenting cells.
 6. The method of claim 1, whereinsaid agent for immunotherapy is at least one agent selected from thegroup consisting of interleukin-2, interleukin-12, tumor necrosisfactor-alpha, and T-cells.
 7. The method of claim 1, wherein saidassessing comprises contacting said tissue sample with an antibody thatspecifically binds to B7-H1 polypeptide.
 8. The method of claim 1,wherein said immuno-inhibitory threshold level is determined byperforming a statistical analysis of the correlation between theproportion of cancer cells and/or tumor-infiltrating leukocytesexpressing B7-H1 in a group of subjects with said cancer and an expectedrisk of death in said group of subjects, and assigning a statisticallysignificant cut-off value for said correlation.
 9. The method accordingto claim 8, wherein the statistical analysis is performed using ascatter plot.